This invention relates to the use of agents that disrupt actin cytoskeletal organization as upregulators of Type III endothelial cell Nitric Oxide Synthase. Further, this invention relates to methods that employ agents that disrupt actin cytoskeletal organization to treat conditions that result from the abnormally low expression and/or activity of endothelial cell Nitric Oxide Synthase in a subject.
Nitric oxide (NO) has been recognized as an unusual messenger molecule with many physiologic roles, in the cardiovascular, neurologic and immune systems (Griffith, T M et al., J Am Coll Cardiol, 1988, 12:797-806). It mediates blood vessel relaxation, neurotransmission and pathogen suppression. NO is produced from the guanidino nitrogen of L-arginine by NO Synthase (Moncada, S and Higgs, E A, Eur J Clin Invest, 1991, 21(4):361-374). In mammals, at least three isoenzymes of NO Synthase have been identified. Two, expressed in neurons (nNOS) and endothelial cells (Type III-ecNOS), are calcium-dependent, whereas the third is calcium-independent and is expressed by macrophages and other cells after induction with cytokines (Type II-iNOS) (Bredt, D S and Snyder, S H, Proc Natl Acad Sci USA, 1990, 87:682-685, Janssens, S P et al., J Biol Chem, 1992, 267:22964, Lyons, C R et al., J Biol Chem, 1992, 267:6370-6374). The various physiological and pathological effects of NO can be explained by its reactivity and different routes of formation and metabolism.
Recent studies suggest that a loss of endothelial-derived NO activity may contribute to the atherogenic process (O""Driscoll, G, et al., Circulation, 1997, 95:1126-1131). For example, endothelial-derived NO inhibits several components of the atherogenic process including monocyte adhesion to the endothelial surface (Tsao, P S et al., Circulation, 1994, 89:2176-2182), platelet aggregation (Radomski, M W, et al., Proc Natl Acad Sci USA, 1990, 87:5193-5197), vascular smooth muscle cell proliferation (Garg, U C and Hassid, A, J Clin Invest, 1989, 83:1774-1777), and vasoconstriction (Tanner, F C et al., Circulation, 1991, 83:2012-2020). In addition, NO can prevent oxidative modification of low-density lipoprotein (LDL) which is a major contributor to atherosclerosis, particularly in its oxidized form (Cox, D A and Cohen, M L, Pharm Rev, 1996, 48:3-19).
It has been shown in the prior art that hypoxia downregulates ecNOS expression and/or activity via decreases in both ecNOS gene transcription and mRNA stability (Liao, J K et al., J Clin Invest, 1995, 96:2661-2666, Shaul, P W et al., Am J Physiol, 1997, 272: L1005-L1012). Thus, ischemia-induced hypoxia may produce deleterious effects, in part, through decreases in ecNOS activity.
HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is the microsomal enzyme that catalyzes the rate limiting reaction in cholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductase inhibitor inhibits HMG-CoA reductase, and as a result inhibits the synthesis of cholesterol. A number of HMG-CoA reductase inhibitors has been used to treat individuals with hypercholesterolemia. Clinical trials with such compounds have shown great reductions of cholesterol levels in hypercholesterolemic patients. Moreover, it has been shown that a reduction in serum cholesterol levels is correlated with improved endothelium-dependent relaxations in atherosclerotic vessels (Treasure, C B et al., N Engl J Med, 1995, 332:481-487). Indeed, one of the earliest recognizable benefits after treatment with HMG-CoA reductase inhibitors is the restoration of endothelium-dependent relaxations or ecNOS activity (supra, Anderson, T J et al., N Engl J Med, 1995, 332:488-493).
Although the mechanism by which HMG-CoA reductase inhibitors restore endothelial function is primarily attributed to the inhibition of hepatic HMG-CoA reductase and the subsequent lowering of serum cholesterol levels, little is known on whether inhibition of endothelial HMG-CoA reductase has additional beneficial effects on endothelial function.
By inhibiting L-mevalonate synthesis, HMG-CoA reductase inhibitors also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) (Goldstein, J L and Brown, M S, Nature, 1990, 343:425-430). The isoprenoids are important lipid attachments for the post-translational modification of variety of proteins, including G-protein and G-protein subunits, Heme-a, nuclear lamins, Ras, and Ras-like proteins, such as Rho, Rab, Rac, Ral or Rap (Goldstein, J L and Brown, M S, supra; Casey, P J, Science, 1995, 268:221-225). The role that isoprenoids play in regulating ecNOS expression, however, is not known.
Pulmonary hypertension is a major cause of morbidity and mortality in individuals exposed to hypoxic conditions (Scherrer, U et al., N Engl J Med, 1996, 334:624-629). Recent studies demonstrate that pulmonary arterial vessels from patients with pulmonary hypertension have impaired release of NO (Giaid, A and Saleh, D, N Engl J Med, 1995, 333:214-221, Shaul, P W, Am J Physiol, 1997, 272: L1005-L1012). Additionally, individuals with pulmonary hypertension demonstrate reduced levels of ecNOS expression in their pulmonary vessels and benefit clinically from inhalation nitric oxide therapy (Roberts, J D et al., N Engl J Med, 1997, 336:605-610, Kouyoumdjian, C et al., J Clin Invest, 1994, 94:578-584). Conversely, mutant mice lacking ecNOS gene or newborn lambs treated with the ecNOS inhibitor, Nw-monomethyl-L-arginine (LNMA), develop progressive elevation of pulmonary arterial pressures and resistance (Steudel, W et al., Circ Res, 1997, 81:34-41, Fineman, J R et al., J Clin Invest, 1994, 93:2675-2683). It has also been shown in the prior art that hypoxia causes pulmonary vasoconstriction via inhibition of endothelial cell nitric oxide synthase (ecNOS) expression and activity (Adnot, S et al., J Clin Invest, 1991, 87:155-162, Liao, J K et al., J Clin Invest, 1995, 96, 2661-2666). Hence, hypoxia-mediated downregulation of ecNOS may lead to the vasoconstrictive and structural changes associated with pulmonary hypertension.
Often cited as the third most frequent cause of death in the developed countries, stroke has been defined as the abrupt impairment of brain function caused by a variety of pathologic changes involving one or several intracranial or extracranial blood vessels. Approximately 80% of all strokes are ischemic strokes, resulting from restricted blood flow. Mutant mice lacking the gene for ecNOS are hypertensive (Huang, P L et al., Nature, 1995, 377:239-242, Steudel, W et al., Circ Res, 1997, 81:34-41) and develop greater intimal smooth muscle proliferation in response to cuff injury. Furthermore, occlusion of the middle cerebral artery results in 21% greater infarct size in xe2x80x9cecNOS knockoutxe2x80x9d mice compared to wildtype mice (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987). These findings suggest that the ecNOS production may play a role in cerebral infarct formation and sizes. Additionally, since most patients with ischemic strokes have average or normal cholesterol levels, little is known on what the potential benefits of HMG-CoA reductase inhibitor administration would be in cerebrovascular events.
There exists a need to identify agents that improve endothelial cell function.
There also exists a need to identify agents that can be used acutely or in a prophylactic manner to treat conditions that result from low levels of endothelial cell Nitric Oxide Synthase.
The invention involves the discovery that agents which disrupt actin cytoskeletal organization can upregulate endothelial cell Nitric Oxide Synthase (Type III) expression. The invention, therefore, is useful whenever it is desirable to restore endothelial cell Nitric Oxide Synthase activity or increase such activity in a cell, tissue or subject, provided the cell or the tissue expresses endothelial cell Nitric Oxide Synthase.
Nitric Oxide Synthase activity is involved in many conditions, including impotence, heart failure, gastric and esophageal motility disorders, kidney disorders such as kidney hypertension and progressive renal disease, insulin deficiency, etc. Individuals with such conditions would benefit from increased endothelial cell Nitric Oxide Synthase activity. It also was known that individuals with pulmonary hypertension demonstrate reduced levels of Nitric Oxide Synthase expression in their pulmonary vessels and benefit clinically from inhalation of Nitric Oxide. The invention therefore is particularly useful for treating pulmonary hypertension. It also has been demonstrated that hypoxia causes an inhibition of endothelial cell Nitric Oxide Synthase activity. The invention therefore is useful for treating subjects with hypoxia-induced conditions. It also has been discovered, surprisingly, that agents which disrupt actin cytoskeletal organization are useful for reducing brain injury that occurs following a stroke.
According to one aspect of the invention, a method is provided for increasing endothelial cell Nitric Oxide Synthase activity in a subject who would benefit from increased endothelial cell Nitric Oxide Synthase activity in a tissue. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount(s) effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. In one important embodiment agents that disrupt actin cytoskeletal organization do not affect cholesterol levels in a subject. In certain embodiments, however, agents that disrupt actin cytoskeletal organization as well as increasing endothelial cell Nitric Oxide Synthase activity in the tissue of a subject can also affect cholesterol levels in the subject. In certain embodiments, the subject is nonhyperlipidimic. In other embodiments the amount is sufficient to increase endothelial cell Nitric Oxide Synthase activity above normal baseline levels established by age-controlled groups, described in greater detail below.
The subject can have a condition characterized by an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is hypoxia-induced. In other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is chemically induced. In still other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is cytokine induced. In certain important embodiments, the subject has pulmonary hypertension or an abnormally elevated risk of pulmonary hypertension. In other important embodiments, the subject has experienced an ischemic stroke or has an abnormally elevated risk of an ischemic stroke. In still other important embodiments, the subject has heart failure or progressive renal disease. In yet other important embodiments, the subject is chronically exposed to hypoxic conditions.
According to any of the foregoing embodiments, the preferred agent that disrupts actin cytoskeletal organization is selected from the group consisting of a myosin light chain kinase inhibitor, a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. In some embodiments the myosin light chain kinase inhibitor is selected from the group consisting of 2,3-butanedione 2-monoxime, 1-(5-iodonaphthalene-1-sulphonyl)-1-hexahydro-1,4-diazepine hydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride. Likewise, in any of the foregoing embodiments, the method can further comprise co-administering an endothelial cell Nitric Oxide Synthase substrate and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. A preferred agent other than an agent that disrupts actin cytoskeletal organization is selected from the group consisting of estrogens and angiotensin-converting enzyme (ACE) inhibitors. The agents may be administered to a subject who has a condition or prophylactically to a subject who has a risk, and more preferably, an abnormally elevated risk, of developing a condition. The inhibitors also may be administered acutely.
According to another aspect of the invention, a method is provided for increasing endothelial cell Nitric Oxide Synthase activity in a subject to treat a condition favorably affected by an increase in endothelial cell Nitric Oxide Synthase activity in a tissue. Such conditions are exemplified above. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. In important embodiments, agents that disrupt actin cytoskeletal organization do not affect cholesterol levels in a subject. In certain embodiments, however, agents that disrupt actin cytoskeletal organization as well as increase endothelial cell Nitric Oxide Synthase activity in the tissue of a subject can also affect cholesterol levels in the subject. In certain embodiments, the subject is nonhyperlipidimic. Important conditions are as described above. Also as described above, the method can involve co-administration of substrates of endothelial cell Nitric Oxide Synthase and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. Preferred compounds are as described above. As above, the agents that disrupt actin cytoskeletal organization with or without the co-administered compounds can be administered, inter alia, acutely or prophylactically.
According to another aspect of the invention, a method is provided for reducing brain injury resulting from stroke. The method involves administering to a subject having an abnormally high risk of an ischemic stroke an agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the brain of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. As above, important embodiments include the agent being selected from the group consisting of a myosin light chain kinase inhibitor, a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. As above, in some embodiments a myosin light chain kinase inhibitor is selected from the group consisting of 2,3-butanedione 2-monoxime, 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride. Also as above, important embodiments include co-administering a substrate of endothelial cell Nitric Oxide Synthase and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. Likewise, important embodiments include prophylactic and acute administration of the agent(s).
According to another aspect of the invention, a method is provided for treating pulmonary hypertension. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase pulmonary endothelial cell Nitric Oxide Synthase activity in the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. Particularly important embodiments are as described above in connection with the methods for treating brain injury. Another important embodiment is administering the agent prophylactically to a subject who has an abnormally elevated risk of developing pulmonary hypertension, including subjects that are chronically exposed to hypoxic conditions.
According to another aspect of the invention, a method for treating heart failure is provided. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase vascular endothelial cell Nitric Oxide Synthase activity in the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. As discussed above, important embodiments include prophylactic and acute administration of the agent(s). Preferred compounds and co-administration schemes are as described above.
According to yet another aspect of the invention, a method is provided for treating progressive renal disease. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase renal endothelial cell Nitric Oxide Synthase activity in the kidney of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. Important embodiments and preferred compounds and schemes of co-administration are as described above in connection with heart failure.
According to another aspect of the invention, a method for increasing blood flow in a tissue of a subject is provided. The method involves administering to a subject in need of such treatment a first agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the first agent is not an agent selected from the group consisting of a rho GTPase function inhibitor and fasudil. In certain embodiments the first agent is not a myosin light chain kinase inhibitor. In other embodiments the first agent is selected from the group consisting of a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. Other important embodiments include co-administering a second agent to the subject with a condition treatable by the second agent in an amount effective to treat the condition, whereby the delivery of the second agent to a tissue of the subject is enhanced as a result of the increased blood flow. In certain embodiments where a second agent is administered, the condition treatable by the second agent does not involve the brain tissue.
The invention also involves the use of agents that disrupt actin cytoskeletal organization in the manufacture of medicaments for treating the above-noted conditions. Important conditions, compounds, etc. are as described above. The invention further involves pharmaceutical preparations that are cocktails of agents that disrupt actin cytoskeletal organization according to the invention [non-rho GTPase function inhibitor(s)]. In certain embodiments, however, the cocktails can include a rho GTPase function inhibitor(s) that disrupts actin cytoskeletal organization together with the non-rho GTPase function inhibitor agent of the invention. The invention also involves pharmaceutical preparations that are cocktails of agents that disrupt actin cytoskeletal organization together with agents other than agents that disrupt actin cytoskeletal organization that also increase ecNOS activity in a cell.
The invention also involves methods for increasing ecNOS activity in a cell by contacting the cell with an effective amount of an agent that disrupts actin cytoskeletal organization (excluding rho GTPase function inhibitors), alone, or together with any of the agents co-administered as described above, or as a cocktail as described above.
In any of the foregoing aspects of the invention the agent can be a non-fasudil agent that disrupts actin cytoskeletal organization.
These and other aspects of the invention are described in greater detail below.